Photodynamic therapy (PDT) is a treatment technique that uses a photosensitizing dye, which localizes at, or near, the treatment site, and when irradiated in the presence of oxygen serves to produce cytotoxic materials, such as singlet oxygen (O.sub.2 (.sup.1 .DELTA..sub.g)) from benign precursors (e.g. (O.sub.2 (.sup.3 .SIGMA..sub.g -)) Other reactive species such as superoxide, hydroperoxyl, or hydroxyl radicals may be involved. At the doses used, neither the light nor the drug has any independent biological effect. In PDT, the photosensitizer acts in a `catalytic` way, since its function is not to react directly with the cellular targets, but to absorb light energy and to transfer it to molecular oxygen, regenerating ground state photosensitizer.
The effectiveness of PDT is due to three additional factors: i) The photosensitive dyes used in PDT must have the ability to localize at the treatment site relative to surrounding tissue. ii) The high reactivity and short lifetime of activated oxygen means that it has a very short range and is unlikely to escape from the cell in which it is produced; cytotoxicity is therefore restricted to the precise region of tissue absorbing light, perhaps down to the cellular level. iii) Developments in lasers and fiber optics allow a beam of intense light to be delivered precisely to many parts of the body.
For reviews of photodynamic therapy, see U.S. Pat. No. 5,252,720 (incorporated by reference herein); Sindelar et al., (1991); Grossweiner, L. I., (1991); Henderson, B. W. and T. J. Dougherty, (1992); and Moan, J. and K. Berg, (1992). In recent years, considerable effort has been devoted to the synthesis and study of new photosensitizers (a review is found in Brown, S. B. and Truscott, T. G., 1993). The development of more effective photochemotherapeutic agents requires the synthesis of compounds which absorb in the spectral region where living tissues are relatively transparent (i.e., 700-1000 nm), have high triplet quantum yields, and are minimally toxic. The present inventors' texaphyrin molecules absorb strongly in the tissue-transparent 730-770 nm range. The photophysical properties of metallotexaphyrins parallel those of the corresponding metalloporphyrins and the diamagnetic complexes sensitize the production of .sup.1 O.sub.2 in high quantum yield The texaphyrins of the present invention, being completely synthetic, can be tuned so as to incorporate desired properties.
The present invention also relates to catalysts for the cleavage of DNA, in particular, photo-induced site-specific cleavage of DNA in a biological system. An effective photocatalyst for PDT and DNA cleavage would have the following properties:
1. Easily available PA1 2. Low intrinsic toxicity PA1 3. Long wavelength absorption PA1 4. Efficient photosensitizer for singlet oxygen production PA1 5. Fair solubility in water PA1 6. Selective uptake in lipophilic tissue such as atheroma or tumor tissue PA1 7. Showing high affinity for enveloped viruses PA1 8. Quick degradation and/or elimination after use PA1 9. Chemically pure and stable PA1 10. Easily subject to synthetic modification PA1 11. Efficient at physiological temperature and pH PA1 12. Specific for certain biological substrates PA1 13. Easy administered to a biological system
The ability to specifically photo-cleave DNA has important implications for the treatment of various diseases; for destruction of viral DNA; for construction of probes for controlling gene expression at the cellular level and for diagnosis; for footprinting analyses, DNA sequencing, chromosome analyses, gene isolation, recombinant DNA manipulations, and for mapping of large genomes and chromosomes; in chemotherapy; and in site-directed mutagenesis. Potential particular applications for this process further include the subsequent recombination of DNA.
Photodynamic cleavage of DNA is known. For example, Praseuth et al., reported cleavage of plasmid DNA by synthetic water-soluble porphyrins with visible light in the presence of oxygen. Fiel, R. J. (1989) also reported the photosensitized strand cleavage and oxidative-reductive strand scission of DNA by iron porphyrins. In another example, Kobayashi et al. reported cleavage of plasmid DNA by sodium pheophorbide (a derivative of chlorophyll) with visible light in the presence of oxygen. Porphyrin-oligonucleotide derivatives were reportedly used to effect sequence specific modifications of DNA substrates followed by cleavage using hot piperidine (Vlassov et al., 1991; Le Doan et al., 1990). The absorption wavelengths for these porphyrin conjugates were below 700 nm, a range that does not penetrate tissue as effectively as longer wavelengths of light.
The use of ultraviolet light with the drug 8-methoxypsoralen to treat psoriasis is well established. Lee et al. relates to the interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA. Crosslinked photoadducts between pyrimidines and psoralen appear to form. This treatment may result in the development of cancerous cells. Furthermore, irradiation at the short wavelength of about 365 nm does not penetrate the body and is therefore only useful on the body surface. Psoralen-based treatments must allow the drug to leave the body before the patient is exposed to visible light or the reaction will continue on the skin surface.
Sequence specific cleavage of DNA has also been reported for dark reactions using oligonucleotides derivatized with metal complexes. Some examples include oligonucleotide-EDTA-Fe complexes (Strobel, D. A. and P. B. Dervan, 1989; Lin, et al., 1989; Dreyer, G. B. and P. B. Dervan, 1985), oligonucleotide-tricationic porphyrins with metal binding appendages (Groves, J. T. and T. P. Farrell, 1989), oligonucleotide-phenanthroline-copper complexes (Chen, C. H. B. and D. S. Sigman, 1988), oligonucleotide-manganese-porphyrins (Meunier, B. et al., 1993), and iron-porphyrins linked to oligonucleotides (Le Doan et al., 1986, 1987).
Limitations of current photosensitive molecules include lack of good tumor selectivity, and the short wavelength of light required to effect the photoexcitation that is prerequisite to photosensitization. Therefore, characteristics sought in new photosensitizers are the retention or enhancement of tumor localization and absorption in the longer wavelength ranges up to about 800 nm as well as non-toxicity, lack of skin photosensitivity, and ease of production in a pure form.
______________________________________ LIST OF ABBREVIATIONS ______________________________________ DCA: Dichloroacetic acid DCC: Dicyclohexylcarbodiimide DMAP: Dimethylaminopyridine DMF: Dimethylformamide DMT: Dimethoxytrityl protecting group DMT-Cl: Dimethoxytrityl chloride EDC: L-Ethyl-3-[3-(dimethylamino)propyl] carbodiimide EDTA: Ethylenediamine tetraacetic acid IPA: Isopropylalcohol NHS: N-hydroxysuccinimide NM: Nanometers pTSA: p-Toluenesulfonic acid monohydrate TEA: Triethylamine TEAB: Triethylammonium bicarbonate TFA: Trifluoroacetic acid TsCl: Tosyl chloride THF: Tetrahydrofuran Txp(txph)(TX): Texaphyrin ______________________________________